Summary: To advance our long-term goal of understanding the regulation of immunoglobulin isotype switching in human B lymphocytes, we have been studying three enhancer regions lying downstream of the human immunoglobulin C-alpha genes to determine how these regions integrate signals from the B cell milieu to regulate Ig expression in conjunction with Ig gene promoters. These three enhancers correspond to DNase I hypersensitivity (HS) sites designated HS3, HS12 and HS4. We previously found that two T cell signals - IL4 and CD40L - inhibit the activity of the HS12 enhancer in transient transfections in the human B cell line CL01 (of germinal center phenotype). We are using ligation-mediated polymerase chain reaction (LMPCR) technology to localize potential binding sites for transcription factors that might regulate enhancer activity. This method has revealed several positions in the human HS12 enhancer that show altered reactivity with dimethyl sulfate (DMS) in vivo in CL01 in comparison to naked DNA, and that are thus candidates for binding motifs of transcription factors. Several regions around consensus NFkB binding sites show CD40L-induced alterations in DMS reactivity; these sites may contribute to regulation by this agent. Recently we have also explored HS3 and HS4 using LMPCR. In HS4 we found altered reactivity around consensus sites for the nuclear proteins NFkB and Ikaros. We have constructed reporter gene plasmids with mutations in these sites, and find that mutation of the Ikaros site leads to increased enhancer activity, consistent with the inhibitory function generally found for this protein. We are continuing to expand our panel of enhancer mutants to study components of the enhancer regions that contribute to the regulation of Ig heavy chain expression. We are also trying to understand the importance of a conserved palindromic structure that flanks the HS12 enhancer. This palindromic structure is conserved in rat and mouse, even though the actual sequence of the palindrome is not conserved. Reporter plasmids that contain or lack the entire palindrome or part of it have been constructed and are being analyzed for enhancer activity in transient and stable transfections. In a collaborative project with the lab of Dr. Richard Hodes of the NCI we are studying the effects of the ATM gene (ataxia telangiectasia-mutated) on immunoglobulin gene expression in vivo and in vitro. Mice carrying defective ATM genes were found to have defects in expression of certain heavy chain isotypes, and we continue to probe the molecular basis for the defect using semiquantitative assays of switch recombination.